Which statement about proteolytic enzymes and their incubation temperatures is true?

• A. Prot K 25-37°C, Trypsin 37°C, Pepsin 37°C, Pronase 25-37°C
• B. Prot K 4°C, Trypsin 60°C, Pepsin 4°C, Pronase 60°C
• C. Prot K 60°C, Trypsin 25°C, Pepsin 60°C, Pronase 4°C
• D. Prot K 0°C, Trypsin 4°C, Pepsin 25°C, Pronase 60°C

Answer: (A)

Proteolytic enzyme digestion in IHC relies on each enzyme having a practical temperature range where it remains active enough to permeabilize tissue without causing excessive damage. Proteinase K is commonly used at a range from room temperature up to 37°C, giving reliable digestion while preserving morphology. Trypsin is typically applied at 37°C because that is around its optimum for proteolytic activity in many protocols. Pepsin is most effective at 37°C under acidic conditions, a common setup in antigen retrieval using this enzyme. Pronase is usually used around room temperature to 37°C, depending on the protocol, to balance digestion with tissue preservation.

Thus, a combination that lists Proteinase K around 25–37°C, Trypsin at 37°C, Pepsin at 37°C, and Pronase around 25–37°C aligns with standard practice. Using temperatures far from these ranges would either slow the enzymes too much or risk denaturing them and harming tissue structure—for example, trying Trypsin at 25°C is suboptimal, or Pepsin at 60°C would be ineffective or damaging, and Pronase at 4°C would be impractically slow.